Influence of the flanking sequences around the core M-CAT motif on its interaction with

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0.51 which confirmed the results obtained by molecular docking simulations together with the idea of 180° rotated binding orientation (Fig. 21C).

To assess the possible biological relevance of the presence of the low affinity binding motifs in the human genome in vivo, the relative TEAD1 occupancy of both orientation binding sites was determined by using chromatin immunoprecipitation (ChIP) analysis followed by qPCR quantification. In agreement with the binding affinities observed in in vitro experiments, the resulting data showed high-level occupancy of the 5’-3’ oriented C-MYC exon, significantly lower occupancy of the C-C-MYC enhancer containing the inverted CAT, and non-significant occupancy of the control region which did not contain the M-CAT motif in any orientation.

In conclusion, the inverted 5’-CCTTA-3’ motif was found to be able to bind TEAD1-DBD with lower affinity than the classical M-CAT both in vivo and in vitro while the surrounding sequence of the core motif also have an influence, although not so significant.

The structural MS experiments confirmed the previously identified regions L1, L2 and H3 as the binding interface, revealed a considerable loss of flexibility occurring upon DNA binding but failed to provide an explanation for the low affinity binding of the inverted M-CAT. MD simulations then revealed (and smFRET experiment subsequently confirmed) that TEAD1-DBD can bind to the inverted motif in 180° rotated orientation while suggesting, that TEAD1-DBD may at first recognize the overall shape of the major groove, which is similar in both orientations, and then the specific amino acid-nucleotide interactions, whose number and strength differ depending on the motif orientation, stabilize the complex. Taken together, the presence of M-CAT sites with widely different affinities in the human genome may provide the basis for possible regulatory mechanisms relying on the actual concentration of a certain transcription factor in the proximity to a gene regulatory region as was described in chapter 1.1.4. and already reported for some other transcription factors^52,188.

4.2.2.

Influence of the flanking sequences around the core M-CAT motif

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originated from regulatory regions of human genes and contained the core ATTCC binding motif either in the classical 5’-3’ or in the inverted 3’-5’ orientation, was prepared. What these constructs differed in were the identity of the two bases on the 5’ side of the M-CAT core motif and one base on its 3’ side, which were selected to correspond to the two most frequent bases in each position as is deposited in the JASPAR database and summarized in Figure 9. Two M-CATs were selected for each combination of flanking bases.

First, the TEAD1-DBD’s ability to form complexes with all the selected dsDNA constructs had to be checked and the dissociation constant of each complex had to be estimated. Since methods usually used for protein-DNA KD determination (such as thermophoresis, fluorescence anisotropy or gel shifts) are tedious and sample consuming or need one of the complex components to be labelled, they are not suitable for evaluation of multiple complexes in short time. Thus, we have tested the potential of native nESI-MS for KD determination using the same six M-CATs with known dissociation constants from Publication IV. For all the six M-CATs the bound fraction was calculated and compared with the expected bound fraction calculated from the known KD. The results (summarised in Figure 22) have shown that, unfortunately, the method could not be used to determine the actual KD since the bound fractions measured by native MS were higher than expected. This could probably be

attributed to the fact, that DNA was in the nESI ion source charged more easily (lower voltage was needed for it to be visible in the spectrum) than the protein because similar effect was, interestingly, already observed in a previous study of ssDNA/dsDNA

equilibria¹⁸⁹. On the other hand, the nESI-MS results followed the

Figure 22: Comparison of calculated bound fractions from nESI-MS experiments and those expected according to the known KD of each complex which was previously determined by fluorescence anisotropy assay.

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same trend as those obtained by fluorescence anisotropy assay, and it was clearly possible to distinguish between the weak binders and strong binders. Therefore, the nESI-MS was proved to be a useful tool for fast differentiation between high affinity and low affinity oligonucleotides with low sample consumption.

The native MS was subsequently utilized to evaluate the complex formation and to sort the series of M-CATs with different bases flanking the core binding motif according to their affinity to TEAD1-DBD. The results, which are summarized in Figure 23, have shown that all the tested CATs were able to form complex with TEAD1-DBD and that all the M-CATs containing the inverted motif belonged to the weak binders, which is in perfect agreement with the results obtained in Publication IV. Moreover, the bound fractions of the two M-CATs with the same flanking base combination were, in most cases, very similar, showing that the identity of the bases flanking the core binding motif might, indeed, be the main reason for the differences in the affinity of these dsDNA constructs to the TEAD1-DBD.

Figure 23: Bound fractions calculated from native MS spectra of complexes of TEAD1-DBD with oligonucleotides with varying bases around the binding motif. Position of the core binding motif is in the labels shown as an underscore while the motif orientation is shown by + (the classical M-CAT) or – (the inverted one). It is clearly possible to differentiate strong binders (uniform fill) from weak binders (squared).

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Following the initial screening, the HDX-MS was employed to further explore the binding interface. The results (Fig. 24) have shown that, similarily to the six M-CATs examined in Publication IV, the highest differences in deuteration rates could be observed in helix H3 and the L2 loop preceding it, thus suggesting, that all M-CATs bind to the same

Figure 24: Comparison of HDX-MS results depicted as number of exchanged deuterium atoms along the sequence for the series of M-CATs differing in the identity of bases flanking the core binding motif (shown as XX_X in the labels where the underscore signifies the position of the core motif). Regions previously identified as responsible for DNA binding are highlighted in red in the structure scheme under the picture. Inverted binding motives are shown as dotted lines and labelled by a minus symbol. The strongest binders exchange less deuterium atoms and therefore are positioned in the bottom of the chart while the weakest binders show a similar pattern as the sample where no DNA was present (TEAD0 - black line).

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region of TEAD1-DBD. However, as was shown in the data published in Publication IV, the degree of protection from deuteraion strongly depends on the affinity of each M-CAT to the protein. Therefore, the results allowed identification of the strongest binders (5'-GCATTCC(T/A)-3') as well as the weakest binder (3'-GCATTCCA-5'). Interestingly, the sequence identified as the strongest binder differs from the most abundant sequence of a TEAD binding site deposited in the JASPAR database in which an A was at the 5’-terminal position instead of a G suggesting that the higher affinity might potentially compensate for the lower abundance of this motif in the human genome¹⁰⁴.

Currently, the actual dissociation constants of each complex are being measured to complement the obtained information and a publication concerning the influence of the flanking sequences around the core M-CAT motif on its interaction with TEAD1-DBD is being prepared.